肥大细胞激活在围术期应激性肠黏膜屏障损伤中的作用及PAR-2/β-arrestin 2通路的调控机制

Gut barrier disruption often results in perioperative multiple organ dysfunction/failure and remains one of serious clinical challenges. Previous studies have shown that mast cell plays a key role in maintaining the integrity of intestinal barrier, moreover, stabilizing mast cell from activation can dramatically attenuate the up-regulations in intestinal permeability, however, the precise mechanism underlying mast cell activation and intestinal permeability remains largely unknown. Our previous study （supported by NSFC, 30901408）found that tryptase, uniquely and mainly released from mast cell degranulation, could directly injury intestinal epithelial cell (IEC-6) through protease activator receptor 2 (PAR-2), and β-arrestin 2 is the scaffold protein in tryptase mediated IEC-6 injury. Based on the above results, we hypothesized that mast cell activation should participate in the process of perioperative gut barrier disruption, further, tryptase should play a central role in increasing intestinal permeability which is regulated by β-arrestin 2. In order to testify the hypothesis, we will confirm the distinct roles of mast cell activation and the underlying mechanism in gut disruption through in vivo studies, furthermore, we will explore the mechanisms by which tryptase/PAR-2 activation triggers gut disruption by specially knocking down or overexpressing PAR-2 or by using PAR-2 knockout mice in vivo, meanwhile, we will observe the effects of β-arrestin 2 on perioperative intestinal disruption and the mechanisms by which β-arrestin 2 regulates tryptase/PAR-2 signal pathway during the process of gut disruption using β-arrestin 2 knockout mice or by knocking down or overexpressing β-arrestin 2 in vitro. The results will provide novel therapeutic benefits against perioperative gut barrier disruption.

肠黏膜屏障受损是导致围术期多脏器损伤/衰竭的重要原因，既往研究发现抑制肥大细胞激活可明显减轻由应激导致的肠黏膜通透性增加，但具体机制不明。我们在国家自然基金资助下（30901408）的前期研究证实类胰蛋白酶（肥大细胞激活释放的主要介质）能通过PAR-2（蛋白酶激活受体-2）途径导致肠黏膜上皮细胞损伤，且β-arrestin 2起“支架”作用。据此，推测手术应激创伤导致的肥大细胞激活与围术期肠黏膜屏障损害有重要的因果关系，释放的类胰蛋白酶发挥了主要作用并受β-arrestin 2调控。为了验证上述假设，本项目研究将在阐明肥大细胞被激活机制的基础上，通过特异性沉默或抑制PAR-2，探讨其在肠黏膜屏障损伤中的作用机制，并通过沉默或敲除β-arrestin 2，探索β-arrestin 2调控类胰蛋白酶/PAR-2在肠黏膜屏障损伤中的作用，以期为围术期肠黏膜屏障损伤的防治提供新的研究思路与治疗靶点

麻醉手术前焦虑应激是患者等候手术期间常见的症状，既往研究发现焦虑与肠黏膜屏障通透性增加密切相关，可能与肥大细胞激活有关，具体机制不明确。据此，本项目首先在体外建立了肠黏膜屏障模型，发现肥大细胞激活能导致肠黏膜屏障受损，进一步发现类胰蛋白酶（肥大细胞激活释放的主要介质）能直接通过PAR-2途径损伤肠黏膜屏障，而ERK不参与其损伤进程。同时，静脉麻醉剂丙泊酚能通过ROS/ Ca2+机制抑制肥大细胞激活脱颗粒，丙泊酚能改善肥大细胞激活导致的肠粘膜屏障功能损伤；但是丙泊酚不能改善tryptase直接导致的肠黏膜屏障功能损伤。进而，在临床研究中发现，术前焦虑评分与肠黏膜通透性密切相关，且是麻醉后脑功能状态改变的危险因素，缓解术前焦虑，能明显降低术后谵妄的发生率。本研究验证了肥大细胞激活在肠粘膜屏障功能破坏中作用及其相关机制，为此类疾病的临床治疗提供了更多证据，本研究证实了丙泊酚通过抑制肥大细胞激活发挥保护肠黏膜屏障功能的作用，为丙泊酚的应用时机掌握提供了更多依据。此外，本研究发现了术前焦虑评分与肠黏膜通透性相关，可能是导致术后谵妄的原因，为后续的研究提供了新的方向。